Expanding the analysis of JUND function and regulation 148

In β cells, JUND regulates a cohort of genes implicated in redox imbalance and

inflammation, and these genes are commonly dysregulated in models of β cell

dysfunction. However, the extent to which each of these genes contributes to β

cell apoptosis is unknown. For example, NOS2, a nitric oxide synthase, has been

implicated in mediating β cell damage in the context of cytokine treatment

(Zumsteg et al., 2000). However, nitric oxide may play beneficial roles in β cells

under certain circumstances by neutralizing other oxidant types (Broniowska et al., 2015). Thus, the induction of several pro-oxidant genes by JUND may lead to complex downstream effects on redox homeostasis based on the types and subcellular locations of the generated oxidants. While we believe it is unlikely that the observed pro-apoptotic role for JUND can be entirely ascribed to one

particular target gene, close scrutiny of these genes may provide new insights into redox imbalance during metabolic stress.

It should also be noted that the oxidative stress assay used in this work

(CellROX) is not specific to any particular oxidant, but rather detects a range of ROS types. This was a useful approach to screen for general oxidative stress levels; however, follow-up studies using more specific assays could provide better insight into which oxidants are upregulated during glucolipotoxicity in a

JUND-dependent manner. For example, dihydroethidium and spin trapping of nitrogen dioxide can be used to specifically detect superoxide and nitric oxide levels, respectively (Pace and Kalyanaraman, 1993; Zhao et al., 2003). These analyses may also shed light on which JUND targets are contributing to oxidative stress during glucolipotoxicity.

Besides its pro-oxidant role, JUND also regulates several pro-inflammatory genes with connections to islet inflammation, including Ccl2, Cxcl1, and Cxcl2. Interestingly, these genes have been linked to poor outcomes for islet

transplantation (Citro et al., 2012; Piemonti et al., 2002), suggesting that

modulation of JUND levels could be a novel approach to advance this clinically relevant application. It is unlikely that the induction of these pro-inflammatory

genes contributed to the phenotypes observed in our in vitro and ex vivo systems

because their main function involves immune cell recruitment. Nevertheless, we cannot rule out the possibility that these genes also have cell-autonomous roles

in β dysfunction. To better assess the relevance of the pro-inflammatory gene

signature, however, it will be important to investigate the benefit of JUND depletion or MEK inhibition using in vivo islet transplantation models.

Given its impact on oxidative stress and apoptosis, the role of JUND in β cell

compensation during insulin resistance should also be studied. This will require

the generation of a conditional null allele for JUND. Crossing this mouse with a β

cell-specific Cre line will allow for the in vivo assessment of JUND function in β

oxidative stress in β cells during a high fat diet challenge, thus improving insulin

secretion and blood glucose homeostasis. However, it is possible that JUND also regulates insulin secretion independent of its effect on oxidative stress and

apoptosis. Thus, a careful and thorough characterization of this in vivo model will be required to assess the impact of JUND deletion on insulin secretion, oxidative stress, cell survival, and proliferation during high fat feeding.

Similar to JUND depletion, I found a significant improvement in islet cell survival during metabolic stress after treatment with the MEK inhibitor trametinib. The application of this finding to in vivo models of diabetes is complicated by the fact that trametinib treatment has been show to improve insulin resistance (Banks et al., 2015). Thus, it will be impossible to discern whether any improvements in insulin secretion or β cell viability seen with systemic trametinib administration

can be attributed to MEK inhibition in β cells or an indirect effect due to reduced

insulin demand. One approach to more specifically study the role of the

MEK/ERK signaling pathway in β cells in vivo would be to generate a mouse

model with a β cell-specific, inducible overexpression of a dominant negative

form of MEK1, which blocks ERK phosphorylation.

The connections between the PCBPs and JUND regulation include the presence of a poly(C) stretch in the JUND 3’UTR, binding of the PCBP members to the JUND mRNA, and loss of PCBPs leading to altered JUND steady-state protein levels. Together, these findings suggest that the post-transcriptional regulation of

JUND is directly mediated by the PCBPs via 3’UTR binding. To further support this model, however, it would be pertinent to show that deletion of the JUND poly(C) motif impairs its post-transcriptional regulation. A common approach to address this question is to generate a reporter gene flanked by the JUND UTRs containing deletions of putative regulatory elements, including the poly(C) motif. Unfortunately, I have been unable to develop a model system to recapitulate the induction of JUND during glucolipotoxicity despite many attempts, thus I could not confidently assess the functionality of the poly(C) motif. Alternatively, genome editing approaches could be used to delete the cytosine-rich sequence element in the endogenous gene. It is unclear whether this would be feasible in Min6 cells as it would require single cell cloning. On the other hand, a mouse model with tissue-specific and inducible deletion of the JUND poly(C) motif could be used to study whether loss of this sequence element abrogates JUND induction in

isolated islets treated with glucolipotoxicity. If so, it would be interesting to see if this deletion also impacts β cell viability during in vivo stress conditions, such as

high fat feeding.

Lastly, I have established that JUND induction during metabolic stress is a conserved process using isolated mouse and human islets. To extend these findings to the pathogenesis of T2D in humans, it will be critical to evaluate JUND levels in pancreatic tissue samples from diabetic patients. This analysis is

typically performed using immunofluorescence staining of pancreatic sections from deceased organ donors. Our ability to perform this experiment is currently

limited by the lack of an antibody to confidently detect JUND levels by staining. Instead, we have relied on Western blot analyses to assess JUND abundance,

including for isolated islets from db/db mice. Similarly, we could compare JUND

levels in isolated islets from diabetic and non-diabetic human organ donors, however, it is possible that the culturing period after islet isolation would provide time for JUND levels to normalize between groups. Thus, it will be advisable to optimize staining conditions for additional antibodies to determine if JUND levels are altered in islets from diabetic individuals.